Screening And Indentification Of Burkholderia Cepacia And Overexpression Of Its Lipase Gene

Biodiesel is one of the most potential ways to solve the current fossil energy crisis. Using lipase as catalyst for biodiesel production has a broad application prospect because it is in line with the country's new strategy for new energy development due to its mild reaction condition, high efficiency and environmental protection. However, expensive price of lipase reduces competition of biodiesel.In this research, Burkholdria cepacia strain G63, whose lipase is highly thermostable and short-chain alcohol tolerant, was directly screened, and was indentified as B. cenocepacia through molecular biological analysis, the produced lipase was stable in pH ranging from 6.0-10.0 and the optimal pH for lipolytic activity was 9.0. The optimal temperature for the lipase was 60℃, and kept fairly stable below 65℃and retained 80% activity after incubation at 60℃for 4 h. The lipase exhibited highly tolerance to a variety of short-chain alcohols for example alcohol, propanol and isopropanol. The above characteristics made it an ideal enzyme for biodiesel production. The highest lipase activity of the wild strain G63 was 12.4 U/mL in shaking flask. To improve lipase expression level, a series of lipase engineering strains were constructed. The expression level of lipase in E. coli BL21 (DE3) was 50 mg/g cell-wet weight, the highest hydrlysis activity toward olive oil in homologous combinant B. cenocepacia was 32.7 U/mL, and the highest hydrolysis activity of pNPP in heterogeneous combinant Pichia pastoris GS115 was 184.3 U/mL. These results demonstrated that strategies of gene modification and overexpression for lipase of G63 were appropriate.The main work and innovations for the research are listed below.1. Directly screening B. cepacia strain by TB-TA plate. Positive screening rate of this method reached nearly 100%, which was appropriate for large scale screening. G63 was indentified as B. cenocepacia by Haelll-recA RFLP (restriction fragment length polymorphism) and genomovar specific PCR. This was the first lipase-producing B. cepacia strain indentified to genomovar level.2. Genes of lipase and foldase were coloned and overexpressed in E. coli, respectively. Based on the result of signal sequence prediction by informatics, we removed the signal sequence from the lipase gene. Then, the obtained gene without signal sequence was expressed in E. coli pet system. However, lipase was expressed in inclusion body. Lipase inclusion bodies of high purity were prepared by sonication and sodium deoxycholate treatment. The measured lipase expression level in E. coli was 50 mg/g cell-wet weight. Interestingly, foldase, whose N-terminal 70 amino acid residues were cut off according to the prediction result of the protein transmembrane region, was expressed in soluble form in the E. coli pet system. The foldase was purified by Ni-NTA chromatography and used for later lipase refolding.3. Lipase refolding in vitro was detailedly examined. The refolding results confirmed that the efficiency of foldase-assisted refolding was much better than that of the dilution refolding. After detailed studies on folase-lipase molar ratio, refolding pH and refolding time, the optimal refolding conditions were pH7.2, foldase-lipase molar ratio 1:1 and refolding time 12 h, under which conditions, the highest specific activity of the refolded lipase was up to 473.8 U/mg.4. T7 protein expression system was for the first time introduced into B. cenocepacia, by which the lipase was homologously overexpressed. Using B. cenocepacia G63 as a host, T7 RNA polymerase was fistly integrated into its genome and was controlled by lipase promoter. Then, lipase gene controlled by T7 promoter was electroporated into T7 RNA polymerase containing strain. Consequently, the lipase was over-expressed. The whole lipase expression system was composed of T7 recombination strain and expression vetors. T7 recombination strain was constructed by suicide vetor pJQ200SK. Regions of 500 bp before and after lipase operon were coloned and then flanked T7 RNA polymerase gene. T7 RNA polymerase with 500 bp flanking region was inserted into pJQ200SK. Through three parental mating, the modified suicide vector was introduced into G63. After homologous recombination between genome and suicide vector, T7 RNA polymerase gene was integrated into B. cenocepacia genome and resulted in T7 recombination strain. On the other hand, lipase expression vectors with T7 promoter were constructed into four types:Vectors pBBR221ip, pBBR221iplif with lipase native signal sequence, and pBBR22△lip, pBBR22△liplif with pelB signal sequence. The above vectors were respectively electroporated into T7 recombination strains and obtained four lipase expression strains. This is the first report by employing T7 protein expression system to construct a homologous combinant B. cenocepacia lipase strain.5. Active expression of B. cenocepacia lipase in Pichia was preliminarily achieved. Based on bioinformatics analysis, we modified and optimized B. cenocepacia lipase gene by overlap PCR to be appropriately expressed in P. pastoris GS115. Using vectors pGAPZa and pPIC9K, constitutive expression and inducible expression GS115 strains were constructed. After fermentation, the highest hydrolysis activity of pNPP was 184.3 U/mL,129.5 U/mL, respectively, which indicatd that GAP promoter was more appropriate for B. cenocepacia lipase expression in Pichia than AOX1 promoter. Enzymatic properties studies showed recombinant lipase expressed in GS115 was identical to the wild type and can also satisfy the needs of industrial application.